Transfection Of PEGFP-N1-wt-p53 Into Bladder Cancer T24 Cells By Ultrasound Combined With Microbubbles

Objective:(1)The parameters of ultrasonic intensity,microbubble/cell suspension ratio,duty ratio and irradiation time were optimized by orthogonal design to determine the effect of different ultrasonic parameters on the permeability of bladder cancer T24 cell membrane,which laid an experimental foundation for the infection of bladder cancer cells by ultrasound combined with microbubble gene.(2)Bladder cancer T24 cells were transfected with p EGFP-N1-wt-p53 gene mediated by ultrasound combined with microbubbles,and their transfection efficiency and apoptosis were detected.Methods:(1)Ultrasonic sound intensity,microbubble/cell suspension ratio,duty ratio and irradiation time were set as the four factors in orthogonal test design.Four levels were set for each factor,namely ultrasonic sound intensity:0.4 W/cm~2,0.6 W/cm~2,0.8 W/cm~2and 1.0W/cm~2,microbubble/cell suspension ratio:10%,20%,30%and 40%,duty ratio:10%,20%,30%and 40%,irradiation time:1 min,2 min,3 min and 4 min.Orthogonal test design table was generated according to four factors and four levels.A total of 16experimental groups were irradiated by ultrasound parameters according to orthogonal test design table.After irradiation,fluorescence microscope was used to detect the positive rate of FD40S staining.(2)Bladder cancer T24 cells were transfected with p EGFP-N1-wt-p53 gene,and the gene transfection rate was significantly increased in the US+MB+Lip2000 control,US+MB,Lip2000,US+Lip2000,and MB+Lip2000 groups(P<0.01);the gene transfection rate was increased in the US+Lip2000 group compared with the Lip2000 group(P<0.01).The apoptosis rate was significantly increased in the US+MB+Lip2000 control,US+MB,Lip2000,US+Lip2000,and MB+Lip2000 groups(P<0.01);the apoptosis rate was increased in the MB+Lip2000 and US+Lip2000 groups compared with the Lip2000group(P<0.01).Results:(1)The effects of different factors on cell membrane permeability from strong to weak were as follows:ultrasonic sound intensity>microbubble/cell suspension volume>duty cycle>irradiation time;The degree of influence of different levels of each factor from strong to weak was as follows:ultrasonic sound intensity:0.6 W/cm~2,0.4 W/cm~2,1.0W/cm~2,0.8 W/cm~2;irradiation time:1 min,2 min,3 min,4 min;duty cycle:40%,10%,20%,30%;microbubble/cell suspension volume:10%,20%,30%,40%.(2)Bladder cancer T24 cells were transfected with p EGFP-N1-wt-p53 gene,and the gene transfection rate was significantly increased in the US+MB+Lip2000 control,US+MB,Lip2000,US+Lip2000,and MB+Lip2000 groups(P<0.01);the gene transfection rate was increased in the US+Lip2000 group compared with the Lip2000 group(P<0.01).The apoptosis rate was significantly increased in the US+MB+Lip2000 control,US+ MB,Lip2000,US+Lip2000,and MB+Lip2000 groups(P<0.01);the apoptosis rate was increased in the MB+Lip2000 and US+Lip2000 groups compared with the Lip2000 group(P<0.01).Conclusion:(1)Different ultrasound parameters had different effects on the permeability of bladder cancer T24 cell membrane,and the optimal combination of their parameters was:ultrasound sound intensity:ultrasound sound intensity:0.6 W/cm~2,irradiation time 1min,duty cycle 40%,and microbubble/cell suspension volume 10%.(2)Ultrasound combined with microbubbles liposome can effectively improve the efficiency of p EGFP-N1-wt-p53 gene transfection into bladder cancer T24 cells,thereby promoting bladder cancer T24 cell apoptosis and providing a new method for the treatment of bladder cancer.